Method for detecting nucleosomes containing nucleotides

ABSTRACT

The invention relates to a method for detecting and measuring the presence of mono-nucleosomes and oligo-nucleosomes and nucleosomes that contain particular nucleotides and the use of such measurements for the detection and diagnosis of disease. The invention also relates to a method of identifying nucleosome associated nucleotide biomarkers for the detection and diagnosis of disease and to biomarkers identified by said method.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a U.S. Continuation Application under 35 U.S.C. §111 which claims the benefit of priority to U.S. National StageApplication No. 14/239,783 under 35 U.S.C. § 371, filed Mar. 27, 2014,which claims the benefit of priority to International Patent ApplicationNo. PCT/GB2012/052128 with International Filing Date of Aug. 31, 2012,which claims the benefit of priority to U.S. Provisional PatentApplication No. 61/530,295, filed Sep. 1, 2011, and to Great BritainPatent Application No. 1115095.0, filed Sep. 1, 2011, each of which ishereby incorporated by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to a method for detecting and measuring thepresence of mono-nucleosomes and oligo-nucleosomes and nucleosomes thatcontain particular nucleotides and the use of such measurements for thedetection and diagnosis of disease. The invention also relates to amethod of identifying nucleosome associated nucleotide biomarkers forthe detection and diagnosis of disease and to biomarkers identified bysaid method.

BACKGROUND OF THE INVENTION

The human body comprises several hundred cell types. All of these celltypes contain the same genome but have widely different phenotypes anddifferent functions in the body. This phenotypic diversity is due to thedifferential expression of the genome in different cell types. Thecontrol of differential gene expression is not entirely understood butthe basic mechanisms include gene regulation by a number ofinterconnected epigenetic signals associated with the gene, includingcontrol of the chromatin packing as euchromatin or heterochromatin,control of nucleosome positioning and nuclease accessible sites,methylation, hydroxymethylation and other modifications of DNA andvariation in the structure of the nucleosomes around which the DNA iswrapped.

The nucleosome is the basic unit of chromatin structure and consists ofa protein complex of eight highly conserved core histones (comprising apair of each of the histones H2A, H2B, H3 and H4). Around this complexis wrapped approximately 146 base pairs of DNA. Another histone, H1 orH5, acts as a linker and is involved in chromatin compaction. The DNA iswound around consecutive nucleosomes in a structure often said toresemble “beads on a string” and this forms the basic structure of openor euchromatin. In compacted or heterochromatin this string is coiledand super coiled into a closed and complex structure (Herranz andEsteller, 2007).

The structure of nucleosomes can vary by Post TranscriptionalModification (PTM) of histone proteins and by the inclusion of varianthistone proteins. PTM of histone proteins typically occurs on the tailsof the core histones and common modifications include acetylation,methylation or ubiquitination of lysine residues as well as methylationof arginine residues and phosphorylation of serine residues and manyothers. Histone modifications are known to be involved in epigeneticregulation of gene expression (Herranz and Esteller, 2007). Thestructure of the nucleosome can also vary by the inclusion ofalternative histone isoforms or variants which are different gene orsplice products and have different amino acid sequences. Histonevariants can be classed into a number of families which are subdividedinto individual types. The nucleotide sequences of a large number ofhistone variants are known and publicly available for example in theNational Human Genome Research Institute NHGRI Histone DataBase(Mariño-Ramírez, L., Levine, K. M., Morales, M., Zhang, S., Moreland, R.T., Baxevanis, A. D., and Landsman, D. The Histone Database: anintegrated resource for histones and histone fold-containing proteins.Database Vol. 2011. (Submitted) andhttp://genome.nhgri.nih.gov/histones/complete.shtml), the GenBank (NIHgenetic sequence) DataBase, the EMBL Nucleotide Sequence Database andthe DNA Data Bank of Japan (DDBJ).

Normal cell turnover in adult humans involves the creation by celldivision of some 10¹¹ cells daily and the death of a similar number,mainly by apoptosis. During the process of apoptosis, chromatin isbroken down into mononucleosomes and oligonucleosomes which are releasedfrom the cells. Under normal conditions the levels of circulatingnucleosomes found in healthy subjects is reported to be low. Elevatedlevels are found in subjects with a variety of conditions including manycancers, auto-immune diseases, inflammatory conditions, stroke andmyocardial infarction (Holdenreider & Stieber, 2009).

Mononucleosomes and oligonucleosomes can be detected by Enzyme-LinkedImmunoSorbant Assay (ELISA) and several methods have been reported(Salgame et al, 1997; Holdenrieder et al, 2001; van Nieuwenhuijze et al,2003). These assays typically employ an anti-histone antibody (forexample anti-H2B, anti-H3 or anti-H1, H2A, H2B, H3 and H4) as captureantibody and an anti-DNA or anti-H2A-H2B-DNA complex antibody asdetection antibody. Using these assays, workers in the field report thatthe level of nucleosomes in serum is higher (by up to an order ofmagnitude) than in plasma samples taken from the same patients. This isalso true for serum and plasma measurements of DNA made by PCR(Holdenrieder et al, 2005). The reason for this is not known but theauthors speculate that it may be due to additional release of DNA duringthe clotting process. However, we have found that the results ofnucleosome ELISA assays of the current art do not agree with each other.Furthermore, although most circulating DNA in serum or plasma isreported to exist as mono-nucleosomes and oligo-nucleosomes(Holdenrieder et al, 2001), measured levels of nucleosomes and DNA inserum or plasma do not agree well. The correlation coefficient betweenELISA results for circulating cell free nucleosomes levels andcirculating DNA levels as measured by real time PCR (Polymerase ChainReaction) has been reported to be r=0.531 in serum and r=0.350 in plasma(Holdenrieder et al, 2005).

Current nucleosome ELISA methods are used in cell culture, primarily asa method to detect apoptosis (Salgame et al, 1997; Holdenrieder et al,2001; van Nieuwenhuijze et al, 2003), and are also used for themeasurement of circulating cell free nucleosomes in serum and plasma(Holdenrieder et al, 2001). Cell free serum and plasma nucleosome levelsreleased into the circulation by dying cells have been measured by ELISAmethods in studies of a number of different cancers to evaluate theiruse as a potential biomarker (Holdenrieder et al, 2001). Meancirculating nucleosome levels are reported to be high in most, but notall, cancers studied. The highest circulating nucleosome levels wereobserved in lung cancer subjects. The lowest levels were observed inprostate cancer, which were within the normal range of healthy subjects.However, patients with malignant tumours are reported to have serumnucleosome concentrations that varied considerably and some patientswith advanced tumour disease were found to have low circulatingnucleosome levels, within the range measured for healthy subjects(Holdenrieder et al, 2001). Because of this and the variety ofnon-cancer causes of raised nucleosome levels, circulating nucleosomelevels are not used clinically as a biomarker of cancer (Holdenriederand Stieber, 2009). Surprisingly we have shown that many cancer subjectswhose circulating nucleosome levels are low or undetectable as measuredby these nucleosome ELISA methods of the current art, do in fact haveraised levels of circulating cell free nucleosomes. We have designed anddemonstrated novel ELISA methods for nucleosomes that detect nucleosomesnot detected by ELISA methods of the current art.

ELISA methods for the detection of histone PTMs are also known in theart. ELISA methods for PTM detection in free histone proteins (notattached to other histones and DNA in a nucleosome complex) are used forthe detection of PTMs in histones extracted, usually by acid extraction,from cell lysates. An immunoassay for the detection of PTMs incirculating cell free nucleosomes has been reported (Bawden et al,2005). A method for ELISA detection of histone PTMs in purifiednucleosomes directly coated to microtitre wells has recently beenreported (Dai et al, 2011). In this method, nucleosomes obtained bydigestion of chromatin extracts from cultured cells are coated directlyto microtitre wells and reacted with anti-PTM antibodies. It will beclear to those skilled in the art that this method requires relativelypure nucleosome samples and is not suitable for the direct measurementof histone PTMs in complex biological media such as blood, plasma orserum.

A modified chromatin immunoprecipitation (ChIP) method for the detectionof a histone PTM (H3K9Me, histone H3 monomethylated at lysine residueK9) in cell free nucleosomes associated with a particular DNA sequencehas been reported in plasma. The level of sequence specific histonemethylation was reported to be independent of the concentration ofcirculating nucleosomes (Deligezer et al, 2008).

Histone variants (also known as histone isoforms) are known to beepigenetic regulators of gene expression (Herranz and Esteller, 2007).Histone variants have been studied in vivo and in vitro using a varietyof techniques including knock-down studies of the gene encoding aparticular variant (for example using RNAi knock-down), chromatinimmunoprecipitation, stable isotope labeling of amino acids andquantitative mass spectrometry proteomics, immunohistochemistry andWestern Blotting (Whittle et al, 2008; Boulard et al, 2010; Sporn et al,2009; Kapoor et al, 2010; Zee et al, 2010; Hua et al, 2008).

Immunohistochemistry studies of histone variant expression in tissuesamples removed at surgery or by biopsy from subjects diagnosed withlung cancer, breast cancer and melanoma have been reported. Theseimmunohistochemistry studies report that staining of histone macroH2A(mH2A) and H2AZ variants in resected cancer tissue samples may haveprognostic application in these cancers (Sporn et al, 2009, Hua et al,2008, Kapoor et al, 2010). One disadvantage of immunohistochemicalmethods for clinical use is that tissue sample collection is invasiveinvolving surgery or biopsy. Another disadvantage ofimmunohistochemistry methods is that they are unsuited for earlydiagnosis or for screening diagnostics as a reasonable expectation ofthe disease must usually already exist before a biopsy or tissueresection is made. Minimally invasive blood ELISA tests are suitable fora wider range of applications and would overcome these disadvantages andbe preferable for the patient as well as faster, lower cost and morehigh-throughput for the healthcare provider.

However, cell free histone variants in cell free nucleosomes have notbeen measured in blood or other media. No studies on the presence orabsence of histone variants in cell free nucleosomes in blood have beenreported. There are currently no methods for the detection ormeasurement of histone variants in intact cell free nucleosomes nor hasany such measurement been suggested or contemplated

In addition to the epigenetic signaling mediated by nucleosome positionand nucleosome structure (in terms of both constituent histone proteinvariant and PTM structures), control of gene expression in cells is alsomediated by modifications to DNA nucleotides including the cytosinemethylation status of DNA. It has been known in the art for some timethat DNA may be methylated at the 5 position of cytosine nucleotides toform 5-methylcytosine. Methylated DNA in the form of 5-methylcytosine isreported to occur at positions in the DNA sequence where a cytosinenucleotide occurs next to a guanine nucleotide. These positions aretermed “CpG” for shorthand. It is reported that more than 70% of CpGpositions are methylated in vertebrates (Pennings et al, 2005). Regionsof the genome that contain a high proportion of CpG sites are oftentermed “CpG islands”, and approximately 60% of human gene promotersequences are associated with such CpG islands (Rodriguez-Paredes andEsteller, 2011). In active genes these CpG islands are generallyhypomethylated. Methylation of gene promoter sequences is associatedwith stable gene inactivation. DNA methylation also commonly occurs inrepetitive elements including Alu repetitive elements and longinterspersed nucleotide elements (Herranz and Estellar, 2007; Allen etal, 2004).

The involvement of DNA methylation in cancer was reported as early as1983 (Feinberg and Vogelstein, 1983). DNA methylation patterns observedin cancer cells differ from those of healthy cells. Repetitive elements,particularly around pericentromeric areas, are reported to behypomethylated in cancer relative to healthy cells but promoters ofspecific genes have been reported to be hypermethylated in cancer. Thebalance of these two effects is reported to result in global DNAhypomethylation in cancer cells (Rodriguez-Paredes; Esteller, 2007).

Hypermethylation of certain specific genes can be used as a diagnosticbiomarker for cancers. For example a method reported for detection ofhypermethylation of the Septin 9 gene by PCR amplification of DNAextracted from plasma was reported to detect 72% of colon cancers with afalse positive rate of 10% (Grutzmann et al, 2008). The DNA methylationstatus of specific genes or loci is usually detected by selectivebisulphite deamination of cytosine, but not 5-methylcytosine, to uracil,leading to a primary DNA sequence change that can be detected bysequencing or other means (Allen et al, 2004).

Global DNA hypomethylation is a hallmark of cancer cells (Estellar 2007and Hervouet et al, 2010). Global DNA methylation can be studied incells using immunohistochemistry (IHC) techniques. Alternatively the DNAis extracted from the cells for analysis. A number of methods have beenreported for the detection of global methylation in DNA extracted fromcells or other media including restriction digestion andnearest-neighbour analysis, fluorescent assays using chloracetaldehyde,inverse determination by methylation of all CpG sites using DNAmethyltransferase in conjunction with tritium-labeled S-adenosylmethionine to calculate the amount of unmethylated CpG and digestion ofDNA into single nucleotides for analysis by high-performance liquidchromatography, thin-layer chromatography, or liquid chromatographyfollowed by mass spectroscopy. The disadvantages of these methods arethat they are labour intensive and/or require large amounts of goodquality extracted DNA (Allen et al 2004). PCR based methods involvingbisulfite deamination overcome the need for large amounts of DNA butmust amplify specific genome regions, typically repetitive sequences, asindicative of the total genome content of 5-methylcytosine (Allen et al2004). These methods for global DNA methylation measurement have beenused to study DNA extracted from a variety of cells and tissues. Someworkers have studied DNA extracted from white blood cells in whole bloodas this is easier to obtain in a minimally-invasive manner (Moore et al,2008; Ting Hsiung et al, 2007; Mansour et al, 2010). LiquidChromatography with mass spectrometry is considered the gold standardfor global DNA methylation measurement but it is costly, and the DNAmust be digested to the single nucleotide level prior to analysis(Vasser et al, 2009).

Recent methods for the estimation of global DNA methylation includeultra high-pressure liquid chromatography with mass spectrometry ofhydrolysed DNA extracted from tissue (Zhang et al, 2011) and amethylation-specific digital sequencing (MSDS) method (Ogoshi et al2011). A classical competitive immunoassay for global DNA methylation(as well as a similar assay for global 5-hydroxymethylcytosinemethylation) has been described. In this method DNA extracted from cellsor tissues is added to a microtitre well coated with a 5-methylatedcytidine conjugate, an anti-5-methylcytidine antibody is added and thedistribution of antibody binding between the coated 5-methylcytidineconjugate and the methylated DNA in the extracted sample is compared tothat of known standards to estimate the global DNA methylation levelpresent in the sample (Cell Biolabs, 2011). In another immunoassay likemethod, DNA extracted from tissues or from plasma or serum samples iscoated to a microtitre well and methylated DNA is detected using ananti-5-methylcytosine antibody (Vasser, et al, 2009; Epigentek, 2009). Adisadvantage of these methods is that they require extraction of DNAinvolving the denaturation and removal of all nucleosome and chromatinstructure from the DNA. They thus cannot measure nucleosome boundnucleotides and are not suited for example; for the direct measurementof global DNA methylation in biological fluids such as tissue lysate,blood, plasma or serum without a DNA extraction step.

5-hydroxymethyl modification of cytosine bases in DNA has also beenreported. The role of 5-hydroxymethylation is not yet well understoodbut it appears to be involved in gene regulation (Stroud et al, 2011).

Current methods for the detection of global DNA methylation involveextraction or purification of the DNA and are not suitable for rapid,high throughput, low cost, minimally-invasive diagnostic methods.Similarly, analysis of DNA for other modified or unusual bases (forexample uracil, inosine, xanthine, hypoxanthine) can only beinvestigated by the analysis of substantially pure or extracted DNA.Such analysis cannot be carried out directly in complex biological mediasuch as tissue lysate, blood, plasma or serum.

Cell free nucleosomes containing 5-methylcytosine or any otherparticular nucleotides or modified nucleotides have not been measured inblood or any other media. No studies on the presence or absence of cellfree nucleosomes containing particular nucleotides in blood have beenreported. Assays for cell free nucleosomes containing particularnucleotides have not been suggested or contemplated. There are currentlyno methods for the detection or measurement of cell free nucleosomeassociated nucleotides.

We now report simple immunoassay methods for the direct estimation ofthe nucleosome associated nucleotides including for example,5-methylcytosine and 5-hydroxymethylcytosine, in biological sampleswithout extraction. Surprisingly we have shown that nucleosomeassociated nucleotides can be detected in blood samples in whichnucleosomes are not detected by ELISA methods of the current art.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a cellfree nucleosome comprising a DNA base, nucleotide or nucleoside for useas a biomarker for the diagnosis of cancer, cardiomyopathy, systemiclupus erythematosus, colitis, chronic obstructive pulmonary disorder,Crohn's disease and rheumatoid arthritis.

According to a second aspect of the invention there is provided a methodfor detecting the presence of a nucleosome containing a DNA base,nucleotide or nucleoside in a sample which comprises the steps of:

-   -   (i) contacting the sample with a binding agent which binds to        the DNA base, nucleotide or nucleoside;    -   (ii) detecting or quantifying the binding of said binding agent        to the DNA base, nucleotide or nucleoside in the sample; and    -   (iii) using the presence or degree of such binding as a measure        of the presence of nucleosomes containing the DNA base,        nucleotide or nucleoside in the sample.

According to a third aspect of the invention there is provided a methodfor detecting the presence of a nucleosome containing a DNA base,nucleotide or nucleoside in a sample which comprises the steps of:

-   -   (i) contacting the sample with a first binding agent which binds        to nucleosomes;    -   (ii) contacting the nucleosomes or sample with a second binding        agent which binds to the DNA base, nucleotide or nucleoside;    -   (iii) detecting or quantifying the binding of said second        binding agent to the DNA base, nucleotide or nucleoside in the        sample; and    -   (iv) using the presence or degree of such binding as a measure        of the presence of nucleosomes containing the DNA base,        nucleotide or nucleoside in the sample.

According to a fourth aspect of the invention there is provided a methodfor detecting the presence of a nucleosome containing a DNA base,nucleotide or nucleoside in a sample which comprises the steps of:

-   -   (i) contacting the sample with a first binding agent which binds        to the DNA base, nucleotide or nucleoside;    -   (ii) contacting the nucleosomes or sample with a second binding        agent which binds to nucleosomes;    -   (iii) detecting or quantifying the binding of said second        binding agent to nucleosomes in the sample; and    -   (iv) using the presence or degree of such binding as a measure        of the presence of nucleosomes containing the DNA base,        nucleotide or nucleoside in the sample.

According to a further aspect of the invention there is provided amethod for detecting the presence of a nucleosome containing a DNA base,nucleotide or nucleoside in a cell which comprises the steps of:

-   -   (i) isolating chromatin from a cell;    -   (ii) digesting, sonicating or otherwise breaking down the        chromatin to form mono-nucleosomes and/or oligo-nucleosomes; and    -   (iii) detecting or measuring the presence of the DNA base,        nucleotide or nucleoside in the said nucleosomes according to a        method of the invention.

According to a further aspect of the invention there is provided amethod for detecting or diagnosing a disease status in an animal or ahuman subject which comprises the steps of:

-   -   (i) detecting or measuring nucleosomes containing a DNA base,        nucleotide or nucleoside in a body fluid of a subject; and    -   (ii) using the nucleosome associated DNA base, nucleotide or        nucleoside level detected to identify the disease status of the        subject.

According to a further aspect of the invention there is provided amethod for assessment of an animal or a human subject for suitabilityfor a medical treatment which comprises the steps of:

-   -   (i) detecting or measuring nucleosomes containing a DNA base,        nucleotide or nucleoside in a body fluid of the subject; and    -   (ii) using the nucleosome associated DNA base, nucleotide or        nucleoside level detected as a parameter for selection of a        suitable treatment for the subject.

According to a further aspect of the invention there is provided amethod for monitoring a treatment of an animal or a human subject whichcomprises the steps of:

-   -   (i) detecting or measuring nucleosomes containing a DNA base,        nucleotide or nucleoside in a body fluid of the subject;    -   (ii) repeating the detection or measurement of nucleosomes        containing a DNA base, nucleotide or nucleoside in a body fluid        of the subject on one or more occasions; and    -   (iii) using any changes in the nucleosome associated DNA base,        nucleotide or nucleoside level detected as a parameter for any        changes in the condition of the subject.

According to a further aspect of the invention there is provided amethod for identifying a DNA base, nucleotide or nucleoside biomarkerfor detecting or diagnosing a disease status in an animal or a humansubject which comprises the steps of:

-   -   (i) detecting or measuring nucleosomes containing the DNA base,        nucleotide or nucleoside in a body fluid of the subject;    -   (ii) detecting or measuring nucleosomes containing the DNA base,        nucleotide or nucleoside in a body fluid of a healthy subject or        a control subject; and    -   (iii) using the difference between the levels detected in        diseased and control subjects to identify whether a DNA base,        nucleotide or nucleoside is useful as a biomarker for the        disease status.

According to a further aspect of the invention there is provided abiomarker identified by said method of the invention.

According to a further aspect of the invention there is provided a kitfor the detection of a nucleosome associated DNA base, nucleotide ornucleoside which comprises a ligand or binder specific for the DNA base,nucleotide or nucleoside or component part thereof, or astructural/shape mimic of the DNA base, nucleotide or nucleoside orcomponent part thereof, together with instructions for use of the kit.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. ELISA dose response curve for the detection of 5-methylcytosinemethylated DNA in cell free nucleosomes in cross-linked digestedchromatin extracted from MCF7 cells diluted into calf serum.

FIG. 2. ELISA dose response curve for the detection of5-hydroxymethylcytosine methylated DNA in cell free nucleosomes incross-linked digested chromatin extracted from A375 cells diluted intocalf serum.

FIG. 3. Nucleosome levels detected for serum and EDTA plasma samplestaken from 20 healthy volunteers using nucleosome ELISA methods of thecurrent art.

FIG. 4. Cell-free nucleosome associated levels of histone variantmH2A1.1 detected for serum and EDTA plasma samples taken from 20 healthyvolunteers.

FIG. 5. Cell-free nucleosome associated levels of histone variant mH2A2detected for serum and EDTA plasma samples taken from 20 healthyvolunteers.

FIG. 6. Cell-free nucleosome associated levels of histone variant H2AZdetected for serum and EDTA plasma samples taken from 20 healthyvolunteers.

FIG. 7. Cell-free nucleosome associated levels of histone modificationP-H2AX(Ser139) detected for serum and EDTA plasma samples taken from 20healthy volunteers.

FIG. 8. Cell-free nucleosome associated levels of 5-methylcytosinemethylated DNA detected for serum and EDTA plasma samples taken from 20healthy volunteers using the ELISA of the invention.

FIG. 9. Cell-free nucleosome associated levels of5-hydroxymethylcytosine methylated DNA detected for serum samples takenfrom 20 healthy volunteers using the ELISA of the invention.

FIG. 10. Cell-free nucleosome associated levels of nucleotides and typesof histones detected for EDTA plasma samples taken from 3 colon cancersubjects.

FIG. 11. Cell-free nucleosome associated levels of nucleotides and typesof histones detected for EDTA plasma samples taken from 13 lung cancersubjects.

FIG. 12. Cell-free nucleosome associated levels of nucleotides and typesof histones detected for EDTA plasma samples taken from 2 pancreaticcancer subjects.

FIG. 13. Cell-free nucleosome associated levels of nucleotides and typesof histones detected for EDTA plasma samples taken from 1 oral cancersubject.

FIG. 14. Cell-free nucleosome associated levels of nucleotides and typesof histones detected for EDTA plasma samples taken from 4 differentcancer diseases normalised as a proportion of nucleosome associated5-methylcytosine methylated DNA levels detected using ELISA methods ofthe invention. Normalised levels for a sample containing nucleosomesfrom healthy volunteers produced by the method of *Holdenrieder et al2001 is shown for comparison (mH2A2 and 5-hydroxymethylcytosine were notmeasured for this sample).

FIG. 15. Cell free nucleosome associated levels of 5-methylcytosine(5mc), mH2A1.1, H2AZ and P-H2AX(Ser139) detected in EDTA plasma, citrateplasma and heparin plasma samples taken from healthy volunteers usingthe ELISA method of the invention.

FIG. 16. Cell free nucleosome associated 5-methylcytosine levelsdetected for serum samples taken from 3 healthy volunteers and 10 coloncancer subjects detected using the ELISA method of the invention.

FIG. 17. Cell free nucleosome associated 5-methylcytosine levelsdetected for EDTA plasma samples taken from 13 healthy volunteers and 55cancer patients. The cut-off points defined as the mean value of thehealthy samples plus one or two standard deviations in the mean areshown.

FIG. 18. Cell free nucleosome associated 5-methylcytosine levelsdetected for EDTA plasma samples taken from 10 healthy volunteers and 61cancer patients. The cut-off point defined as the mean value of thehealthy samples plus two standard deviations in the mean is shown.

FIG. 19. Cell free nucleosome associated 5-methylcytosine levelsdetected for EDTA plasma samples taken from lung and colon cancerpatients with increasing tumour size, stage and nodal development ofdisease.

FIG. 20. Mean cell-free nucleosome associated levels of nucleotides andtypes of histones detected using ELISA methods of the invention for EDTAplasma samples taken from 10 different cancer diseases normalised as aproportion of nucleosome associated 5-methylcytosine (5mc) methylatedDNA levels and expressed relative to the mean proportions found in 11healthy subjects.

FIG. 21. Mean cell-free nucleosome associated levels of nucleotides andtypes of histones detected using ELISA methods of the invention for EDTAplasma samples taken from 2 cardiomyopathy patients, 10 systemic lupuserythematosus (lupus) patients, 12 ulcerative colitis patients, 10chronic obstructive pulmonary disease (COPD) patients, 8 Crohn's diseasepatients and 10 rheumatoid arthritis (RA) patients normalised as aproportion of nucleosome associated 5-methylcytosine (5mc) methylatedDNA levels and expressed relative to the mean proportions found in 11healthy subjects.

DETAILED DESCRIPTION OF THE INVENTION

According to a first aspect of the invention there is provided a cellfree nucleosome comprising a DNA base, nucleotide or nucleoside for useas a biomarker for the diagnosis of cancer, cardiomyopathy, systemiclupus erythematosus, colitis, chronic obstructive pulmonary disorder,Crohn's disease and rheumatoid arthritis.

In one embodiment, the nucleosome is a mononucleosome oroligonucleosome.

According to one particular aspect of the invention which may bementioned, there is provided the use of a DNA base, nucleotide ornucleoside as a biomarker for the diagnosis of cancer.

In one embodiment, the cancer is a cancer of the bladder, breast, colon,cervix, esophagus, kidney, large intestine, lung, oral cavity, ovary,pancreas, prostate, rectum, skin or stomach. In one particularembodiment which may be mentioned, the cancer is a cancer of the colon,lung, oral cavity or pancreas.

We have developed ELISA tests for the detection and measurement ofnucleosomes containing the DNA bases 5-methylcytosine and5-hydroxymethylcytosine. We have used an anti-histone antibody ascapture antibody for these assays in combination with an appropriatespecific anti-nucleotide antibody. We have used the assays to show thatnucleosomes containing specific nucleotides can be measured in bloodsamples taken from subjects with cancer and are discriminating for useas non-invasive or minimally invasive biomarkers. The nucleosomeassociated DNA 5-methylcytosine levels, relative to levels of othernucleosome epitopes, detected in serum and plasma samples taken fromdiseased subjects differed from those detected in samples from healthysubjects. In addition the pattern of levels of the nucleotides detectedin nucleosomes in samples taken from subjects with different diseaseswas found to differ such that a differential diagnosis of disease waspossible, particularly when the nucleosome associated nucleotidepatterns were examined in combination with the patterns determined fornucleosomes containing different histone variants and histonemodifications. It will be clear to those skilled in the art thatinclusion of tests for nucleosomes containing different or additionalnucleotides would be likely to improve the discrimination ofdifferential diagnosis using such patterns.

To investigate levels of nucleosomes found in healthy subjects using themethods of the current art we measured nucleosomes in serum and plasmasamples, taken from the 20 healthy subjects. Both methods of the currentart produced higher signals in serum samples taken from healthy subjectsthan in plasma samples. The results are shown in FIG. 3. This isconsistent with published data that nucleosome levels are higher inserum than plasma (*Holdenrieder et al, 2001).

To investigate levels of nucleosomes found in healthy subjects using themethods of the invention we measured nucleosomes containing the modifiednucleotide 5-methylcytosine in the sera of 20 healthy subjects and inhealthy bovine serum. The serum results were low or undetectable for all20 healthy subjects. We also measured nucleosomes containing themodified nucleotide 5-methylcytosine in EDTA plasma samples, taken fromthe 20 healthy subjects, and, surprisingly, higher signals wereobserved. High levels of cell free nucleosomes containing the modifiednucleotide 5-methylcytosine were detected by methods of the presentinvention in healthy human EDTA plasma but lower levels were detected inhealthy human serum as shown in FIG. 8. FIGS. 4-9 show that similarresults were obtained for other nucleosome structures. This finding isunexpected and different to both the published results (*Holdenrieder etal, 2001) and the results we found for nucleosome ELISA methods of thecurrent art. Thus surprisingly the methods of the invention produceopposite results to methods of the current art for the relative levelsof nucleosomes that occur in serum and EDTA plasma samples.

We investigated whether nucleosome structures are detectable in all ofthe various common types of plasma that can be collected. We found thathigh levels of cell free nucleosome associated 5-methylcytosine weredetectable by the method of the invention in EDTA plasma and, to alesser extent, in citrate plasma taken from healthy subjects, but thatnucleosome associated 5-methylcytosine was low or undetectable overbuffer or horse serum background signals in most (3 of 5) heparin plasmasamples taken from healthy subjects. The results are shown in FIG. 15.To summarise, cell free nucleosomes are found in relatively highconcentrations in most or all EDTA plasma and citrate plasma samplestaken from healthy subjects using the method of the invention, but arelow or absent in a majority of heparin plasma or serum samples takenfrom healthy subjects. It is therefore clear that the precise choice ofsample type will be critical for different applications.

We have shown that sample selection for the detection of cell freenucleosomes containing particular nucleotide structures involves severalparameters. These include the low levels of cell free nucleosomesgenerally present in serum and heparin plasma samples taken from healthysubjects, the higher levels generally present in EDTA and citrate plasmasamples taken from healthy subjects, the recommendation that serumsamples containing cell free nucleosomes should be stabilised by theaddition of EDTA after separation of the serum from the clot(*Holdenreider et al, 2001), and the serum sampling protocol. Otherstabilizing agents (for example protease inhibitors) may also be used.Where possible we used serum samples centrifuged within 1 hour ofvenepuncture after which 10 mM EDTA was added and the sample frozen.

The choice of blood sample type for clinical samples should be made onthe basis of optimal clinical discrimination for the particular test.Following our finding of consistently low nucleosome levels by themethod of the invention in the serum of healthy subjects, we measurednucleosomes containing the nucleotide 5-methylcytosine in serum samplestaken from subjects with cancer. Clinical sensitivity of up to 100% wasobserved as shown in FIG. 16 for colon cancer samples.

We also measured the relative levels of cell free nucleosomes containingthe nucleotides 5-methylcytosine and 5-hydroxymethylcytosine and othernucleosome structures in EDTA plasma samples taken from subjects with avariety of diseases. The levels of cell free nucleosomes are high inEDTA plasma samples taken from both healthy subjects and diseasedsubjects and EDTA plasma samples would therefore seem unlikely to be thebest sample choice for a sensitive discriminator of diseased and healthysubjects. However, we have shown that the levels and the composition ofcirculating cell free nucleosomes, in terms of the relative levels ofnucleosomes containing different nucleotides (as well as othernucleosome structures), varies between diseased and healthy individualsand also between different diseases. We are thus the first to reportboth that (i) high levels of circulating nucleosomes are present in allor most EDTA plasma samples taken from both healthy and diseasedsubjects but this is not true of all blood sample types; and also that(ii) surprisingly, detection of disease and discrimination of diseasetype can none the less be made by analysis of these EDTA plasmanucleosomes on the basis of the levels and structural profile of one ormore of the relative types of nucleosome structures present in theplasma of diseased and healthy subjects.

We measured cell free nucleosomes in EDTA plasma taken from healthysubjects and 117 subjects with a variety of cancer types in twoexperiments consisting of 55 and 62 cancer subjects respectively. Intotal 78% (91 of 117) of cancer samples were correctly identified aspositive for cancer using the method of the invention for nucleosomeassociated 5-methylcytosine using a cut-off level of the mean result forhealthy subjects+2 standard deviations of the mean.

In the first of these 2 experiments we measured cell free nucleosomes inEDTA plasma taken from 13 healthy subjects and 55 subjects with cancerof the stomach, large intestine, rectum, lung (small cell carcinoma andvarious non-small cell carcinomas), breast, ovary, pancreas, prostate,kidney and various oral cancers (oral cavity, palate, pharynx andlarynx). All of the 13 samples from healthy subjects and cancer patientswere positive for nucleosomes. However, the levels detected in samplestaken from cancer subjects were higher than found in samples fromhealthy subjects and the results showed that healthy and cancer subjectscan be discriminated. For example the normal range calculated in ODterms as the mean±2 standard deviations of the mean, for nucleosomeassociated 5-methylcytosine was 0-1.41. Using this cut-off value all 13healthy samples were negative and 30 of the 55 cancer samples werepositive. (an overall clinical sensitivity of 55%) including 38% (3 of8) of stomach, 60% (3 of 5) of large intestinal, 33% (1 of 3) of rectal,33% (2 of 6) small cell lung, 64% (9 of 14) of non-small cell lung, 33%(2 of 6) of breast, 100% (1 of 1) of ovarian, 100% (1 of 1) of pancreas,33% (2 of 6) of prostate, 100% (1 of 1) of kidney and 60% (3 of 5) oforal cancer samples. The results are shown in FIG. 17.

Similarly the normal range for the nucleosome associated H2AZ assay was0-0.95. Using this cut-off level of 0.95; all 13 healthy subjects werenegative for elevated nucleosome H2AZ levels. By contrast a positiveresult for elevated nucleosome H2AZ levels was found for 84% (46 of 55)of cancer samples (an overall clinical sensitivity of 84%) including100% (8 of 8) of stomach 100% (5 of 5) of large intestinal, 67% (2 of 3)of rectal, 83% (5 of 6) of small cell lung, 79% (11 of 14) of non-smallcell lung, 50% (3 of 6) breast, 100% (1 of 1) of ovarian, 100% (1 of 1)of pancreas, 80% (4 of 5) of prostate, 100% (1 of 1) kidney and 100% (5of 5) oral cancer samples.

In one embodiment of the invention a control sample is provided and thecut-off level for the assay to distinguish between positive or negativeresults is defined in relation to the result for the control sample.This could be any proportion equal to or above or below the level of thecontrol sample result. Patient results below this level are considerednegative and patient results above this level are considered positive.There may also be a “grey area” range of patient results very close tothe cut-off level for which the decision is considered indeterminateand/or the test should be repeated.

Similarly for the nucleosome associated mH2A1.1 assay the normal rangewas 0-0.91. Using this cut-off value all 13 healthy samples werenegative and 64% (35 of 55) of cancer samples were positive. For thenucleosome associated P-H2AX(Ser139) assay the normal range was 0-1.08.Using this cut-off value all 13 healthy samples were negative and 60%(33 of 55) of cancer samples were positive. Thus some nucleosome assaysexhibit better clinical sensitivity than others.

In addition, it is possible to use the pattern of nucleosome structuresto improve the clinical utility of the invention. This may be done, forexample, by lowering the cut-off point of the nucleosome associated5-methylcytosine assay to mean+1 standard deviation which gives a rangeof up to 1.01. In this case the number of false negatives is reduced to4 giving an improved clinical sensitivity of 93% (51 of 55) at theexpense of an increase in false positive results for samples taken fromhealthy subjects from 0% to 23% (3 of 13). The results are shown in FIG.17.

Samples found positive for 5-methylcytosine associated nucleosomes, orany nucleosomes, can be interrogated for nucleosome structure profile.The nucleosome profile can be used to distinguish between healthy anddiseased patients as illustrated in FIGS. 20 and 21 where the relativeproportions of various nucleosome structures in diseased patients areexpressed relative to those found in healthy patients and patients withother non-cancer diseases. This shows that investigation of multiplenucleosome structures in a test panel can facilitate better clinicaldiscrimination.

Similarly the diagnostic specificity and/or sensitivity of the inventionmay by increased by combining data from more than one test in the formof ratios. For example use of the nucleosome associatedP-H2AX:5-methylcytosine ratio increases the detection of true positivecancer cases from 55% (30 of 55) for nucleosome associated5-methylcytosine alone, to 67% (37 of 55) at the 2 standard deviationcut-off level whilst maintaining 100% (13 of 13) of negative results forsamples taken from healthy subjects.

We measured the levels of circulating cell free nucleosomes containingtwo different nucleotides in EDTA plasma samples taken from 3 patientswith colon cancer, 13 patients with lung cancer, 2 patients withpancreatic cancer and 1 patient with oral cancer and compared these withthe levels present in blood samples from 20 healthy subjects as well aswith an artificially produced preparation of serum nucleosomes fromhealthy subjects prepared as described in the literature (*Holdenreideret al, 2001). We have also expressed the levels observed in a normalisedform as ratios of the level of nucleosomes containing one particularnucleotide and shown that such ratios or patterns of ratios are usefulfor the diagnosis both of cancer in general and for the differentialdiagnosis of specific cancer types. We also investigated whether thelevel of nucleosome associated 5-methylcytosine varies with diseaseprogression. We observed that the mean level of cell free nucleosomescontaining 5-methylcytosine increases with severity of disease and riseswith increasing spread of disease to lymph nodes. This provides evidencethat the nucleosomes detected are tumour associated.

We also measured the nucleosomes present in these 19 cancer samplesusing two nucleosome ELISA methods of the current art. Of the 19 cancersubjects studied most were found to have low EDTA plasma nucleosomelevels as determined by nucleosome ELISA 1 and 2 of the current art.This result illustrates one reason why the assays of the current art arenot used for routine clinical purposes.

We used ELISA methods of the present invention to measure nucleosomescontaining 5-methylcytosine and 5-hydroxymethylcytosine nucleotides inthe same 19 samples. Surprisingly, high levels of nucleosomes containing5-methylcytosine were detectable in all 19 samples. Thus in oneembodiment the invention provides a novel nucleosome ELISA methodcapable of detecting nucleosomes not detected by nucleosome assays ofthe current art.

We have also measured the levels of nucleosomes containing 3 differenthistone variants and a histone PTM in the same 19 samples taken fromcancer subjects as well as a sample of nucleosomes generated fromhealthy subjects by a method described in the literature (*Holdenriederet al, 2001). We have used these measurements together with thenucleosome associated nucleotide measurements described here, as a panelof the variety of cell free nucleosomes present in biological fluidstaken from subjects with 4 different types of cancers and withnucleosomes generated from healthy subjects. Surprisingly, the patternof nucleosomes found in the 4 types of cancer investigated (lung, colon,pancreatic and oral) were all distinguishable from that found in thenucleosome sample generated from healthy subjects. Furthermore, thedifferent cancer types were also distinguishable from each other basedon the pattern of cell free nucleosomes detectable in the blood ofsubjects. Thus in one embodiment of the invention there is provided amethod for detecting or diagnosing the presence, type, recurrence orseverity of a disease or assessing optimal drug or other treatmentoptions by testing a sample for a panel of different nucleosome epitopesconsisting of two or more measurements of nucleosomes containingdifferent DNA bases or a combination of one or more DNA bases and one ormore histone variants and/or one or more histone modifications and/ormeasurements of nucleosomes per se, or any combination or ratio of anyof these, as an indicator of the health or disease status of a subject.

We similarly used ELISA methods of the invention to detect variabilityin the nucleotide and histone structures of circulating cell freenucleosomes in a variety of cancer and non-cancer diseases and comparedthese to the structure of nucleosomes found in healthy subjects.Nucleosomes were found to be present in all the cancer and non-cancerdiseases investigated and were found to have profiles that differed fromthose of healthy subjects.

We studied EDTA plasma samples taken from 2 cardiomyopathy patients, 10systemic lupus erythematosus (lupus) patients, 12 ulcerative colitispatients, 10 chronic obstructive pulmonary disease (COPD) patients, 8Crohn's disease patients and 10 rheumatoid arthritis (RA) patients andnormalised the levels of various nucleosome structures detected as aproportion of the mean nucleosome associated 5-methylcytosine levels andexpressed the results relative to those found in 11 healthy subjects. Wefound that the diseases were associated with nucleosome structureprofiles that differed from those of healthy or cancer subjects. Thusnucleosome structure profiles can be used as a diagnostic tool for thedetection, prognosis prediction, monitoring and therapeutic efficacyprediction in a wide variety of non-cancer diseases. The results areshown in FIG. 21.

We also studied the variability in structure of cell-free nucleosomes interms of nucleotides and types of histones detected using ELISA methodsof the invention for EDTA plasma samples taken from 55 patients with 10different cancer diseases. The levels of various nucleosome structuresdetected were normalised as a proportion of nucleosome associated5-methylcytosine (5mc) methylated DNA levels and expressed relative tothe mean proportions found in 11 healthy subjects. We found nucleosomespresent in all subjects and nucleosome structure profiles that variedbetween cancer diseases, non-cancer diseases and healthy subjects. Thusnucleosome structure profiles can be used as a diagnostic tool for thedetection, prognosis prediction, monitoring and therapeutic efficacyprediction in cancer and other diseases. The results are shown in FIGS.20 and 21.

As most circulating DNA in serum or plasma is reported to exist asmono-nucleosomes and oligo-nucleosomes (Holdenrieder et al, 2001), itwill be clear to those skilled in the art that methods of the currentinvention can also be employed to detect or measure cell free methylatedDNA per se (as nucleosome associated DNA containing for example;5-methylcytosine or 5-hydroxymethylcytosine) directly in biologicalfluids including blood, serum and plasma. The methods of the inventionthus employed have advantages of simplicity and speed over methods formeasuring methylated DNA of the current art, particularly as extractionof DNA is not involved or required.

It will further be clear that the method of the present invention can beused to detect or measure any nucleic acid or DNA base or nucleic acidanalogue or derivative in nucleosomes. Such bases include, withoutlimitation adenine, thymine, guanine, cytosine, uracil, inosine,xanthine, hypoxanthine, 7,8-dihydro-8-oxo-guanine and any derivatives oranalogues of these. It will be clear to those skilled in the art that acommon nucleotide (for example without limitation; guanine, cytosine,thymine or adenine), will occur in all or most nucleosomes and that themethod of the invention using an antibody to a common nucleotide willprovide a method to bind and detect virtually all nucleosomes in asample. Thus in one embodiment the invention provides a novel method forthe detection of nucleosomes per se in which nucleosomes containing acommon nucleotide are measured as a way of ensuring that all or mostnucleosomes are detected.

In a further embodiment the invention provides a novel method for thedetection of all nucleosome associated DNA in which nucleosomescontaining a common nucleotide are measured as a way of ensuring thatall or most nucleosome bound DNA is detected. Furthermore, measurementof two or more DNA bases will provide the basis for the measurement of aratio of the relative DNA content of those DNA bases. We illustrate suchratios for the relative levels of 5-methylcytosine and5-hydroxymethylcytosine in samples in FIGS. 10-14. Our data show thatthe relative levels of 5-methylcytosine and 5-hydroxymethylcytosinedetectable differs in different types of cancers and may be used todistinguish such cancers. Other similar ratios would also be useful inthe art. For example; by using the present invention to measure anappropriate DNA base (or bases) as a metric for total nucleosome boundDNA and determining the relative level of another base (for example;5-methylcytosine) it will be clear that the method of the invention canbe used to detect the proportion of the DNA which comprises anyparticular base (for example the percentage of DNA which is methylatedin a sample). Thus the methods of the present invention provide a simpleand rapid method for measurement of the percentage DNA content of anybase in a sample. The method can be used quickly and simply in multiplesamples, for example blood samples. The methods of the invention can beused to detect and measure DNA bases in nucleosomes in any sample wheresuch nucleosomes occur including, for example, samples obtained bydigestion of chromatin extracted from cells. It will be clear to thoseskilled in the art that the term nucleotide herein is intended toinclude without limitation purines, pyrimidines or any other nucleicacid bases and similar molecules with or without associated sugars andwith or without phosphorylation and including any analogues, derivativesor mimics of these.

We conclude that the method of the present invention is a successfulmethod for the detection and measurement of nucleosome associated DNAcontaining particular nucleotides, that this method can also be usedsuccessfully as a method for the detection of nucleosomes per se andthat it is a superior method for the detection of nucleosomes per sethan the methods of the current art and that this method can also beused successfully as a method for the direct detection of cell free DNAper se and for the nucleotide composition of cell free DNA per se andthat it is a superior method for the detection of nucleosome associatedDNA and its nucleotide composition than the methods of the current art.The method is rapid, low cost and suitable for use in complex biologicalmedia and fluids. We have demonstrated that the method of the currentinvention can be used to detect nucleosomes and nucleosomes containingmethylated DNA in blood, and that this may be used as a biomarker forcancer. It will be clear to those skilled in the art that a biomarkerpresent in the blood samples taken from cancer patients has value for abroad range of diagnostic and disease screening purposes for cancer andother diseases which are associated with elevated circulatingnucleosomes (Holdenrieder et al, 2001).

To confirm that elevated levels of nucleosomes are not found in healthysubjects using the methods of the invention we measured nucleosomescontaining the nucleotides 5-methylcytosine and 5-hydroxymethylcytosinein the sera of 20 healthy subjects and in healthy bovine serum. Theserum circulating nucleosome results for both ELISA tests of theinvention were low or undetectable for all 20 healthy subjects. We alsoconducted a similar test in plasma samples, taken from the same 20healthy subjects and surprisingly, higher signals were observed. Thisfinding is unexpected and quite different from the results we found fornucleosome ELISA methods of the current art.

The invention has been tested on many cancer and non-cancer diseases andhas been found effective in the detection of all the diseases tested.This includes the detection of prostate cancer cases which is notdetectable by the nucleosome ELISA tests of the current art(Holdenrieder, 2001). It is clear that the invention is effective forthe detection of all or most cancers. It will be clear to those skilledin the art that the clinical performance of the invention may beimproved further by inclusion of further nucleosome structure tests andby examination of the ratios of different nucleosome structures present.

According to one aspect of the invention there is provided a doubleantibody, immunometric or sandwich immunoassay method for detecting andmeasuring cell free nucleosomes containing nucleotides in a sample. Oneembodiment of this aspect is an immunoassay which comprises the stepsof:

-   -   (i) contacting the sample which may contain nucleosomes with a        first antibody or other binder which binds to nucleosomes;    -   (ii) contacting the nucleosomes or sample with a second antibody        or other binder which binds to a nucleotide;    -   (iii) detecting and/or quantifying the binding of said second        antibody or other binder to a nucleotide in the sample; and    -   (iv) using the presence or degree of such binding as a measure        of the presence of a nucleosome associated nucleotide in the        sample.

According to a second embodiment there is provided a method fordetecting and measuring cell free nucleosomes containing nucleotides ina sample by an immunometric immunoassay which comprises the steps of:

-   -   (i) contacting the sample which may contain nucleosomes with a        first antibody or other binder which binds to a nucleotide;    -   (ii) contacting the nucleosomes or sample with a second antibody        or other binder which binds to nucleosomes;    -   (iii) detecting and/or quantifying the binding of said second        antibody or other binder to nucleosomes in the sample; and    -   (iv) using the presence or degree of such binding as a measure        of the presence of a nucleosome associated nucleotide in the        sample.

A variety of antibodies or other binders may be employed in theinvention as a binder which binds to nucleosomes. These include bindersdirected to bind to epitopes that occur in intact nucleosomes and not infree histones (for example; an epitope found at the junction between twohistones in a nucleosome) and also binders directed to any nucleosomecomponent including common nucleosome protein, histone or nucleic acidepitopes.

It will be clear to those skilled in the art that the methods of theinvention described include a variety of embodiments including biosensortype assays and label-free assays of the type marketed for example byForteBio Incorporated of USA. Immunometric immunoassays employ anantibody (or other binder) to bind the analyte. The analyte thus boundis detected as a direct measure of its level or concentration in theoriginal test sample. In contrast “competitive” immunoassays often use amuch smaller amount of antibody (or other binder) to bind a proportionof the analyte and a labelled analyte (or analyte analogue) preparationis employed to distribute between the bound and free analyte fractions(with the sample analyte). The amount of bound labelled analyte ismeasured as an indirect measure of the analyte concentration in theoriginal sample. In a variation of “competitive” immunoassay design alabelled antibody is employed, together with a solid phase analyte (oranalyte analogue) preparation. The binding of the labelled antibody isdistributed between the sample analyte and the solid phase analyte (oranalyte analogue). The amount of antibody bound to the solid phaseanalyte (or analyte analogue) preparation is used as an indirect measureof the analyte concentration of the sample.

According to a third embodiment of the invention there is provided amethod for detecting and measuring a nucleotide, including a nucleosomeassociated nucleotide, in a sample by a label-free immunometricimmunoassay which comprises the steps of:

-   -   (i) contacting the sample with an antibody or other binder which        binds to a nucleotide;    -   (ii) detecting and/or quantifying the binding of said antibody        or other binder to a nucleotide in the sample; and    -   (iii) using the presence or degree of such binding as a measure        of the presence of a nucleotide in the sample.

According to a fourth embodiment of the invention there is provided amethod for detecting and measuring a nucleotide, including a nucleosomeassociated nucleotide, in a sample by a competitive immunoassay whichcomprises the steps of:

-   -   (i) contacting the sample with an antibody or other binder which        binds to a nucleotide;    -   (ii) detecting and/or quantifying the binding of said antibody        or other binder to a nucleotide in the sample; and    -   (iii) using the presence or degree of such binding as a measure        of the presence of a nucleotide in the sample.

It will be clear to those skilled in the art that these immunoassaymethods of the invention measure nucleotides and nucleosome associatednucleotides directly without any requirement for extraction of DNA. Incontrast, nucleotide immunoassay methods of the current art detect(non-nucleosome associated) nucleotides after extraction of DNA from asample. The methods of the invention have advantages of speed,simplicity and suitability for direct measurements in complex biologicalsamples including blood or its derivatives.

According to a fifth embodiment of the invention there is provided amethod for detecting the proportion of cell free DNA that comprises aparticular nucleotide in a sample comprising the steps of:

-   -   (i) detecting or measuring the level of cell free DNA in a        sample;    -   (ii) detecting or measuring the level of a nucleosome associated        nucleotide according to a method of the invention; and    -   (iii) using the two measurements to determine the proportion of        DNA that comprises the nucleotide.

According to one embodiment of this aspect of the invention; both thecell free DNA level in the sample and the nucleotide of interest aremeasured using the method of the invention. In another embodiment thenucleotide of interest is a methylated cytosine nucleotide and theproportion of the DNA that comprises the nucleotide provides a measureof global DNA methylation.

We have shown that the detection and measurement of nucleosomescontaining nucleotides in the blood taken from subjects can be used as adiagnostic method to identify subjects with cancer and to differentiatethem from healthy subjects. Furthermore we have shown that the patternsof nucleosomes containing a panel of different nucleotides, histonevariants and histone PTMs can be used to distinguish between differentcancers. It will be clear to those skilled in the art that this providesa cancer blood test that will detect cancer in subjects and can be usedto distinguish between cancer types in cancer positive subjects.According to a further aspect of the invention there is provided amethod for detecting or diagnosing the presence of a disease bymeasuring or detecting the presence and/or the level or concentration ofcell free nucleosomes containing a nucleotide in a body fluid, and usingthe detected level as a biomarker of the disease status of a subjectincluding, without limitation, a clinical diagnosis of a disease, adifferential diagnosis of disease type or subtype, or a diseaseprognosis, or a disease relapse, or a diagnosis of subjectsusceptibility to treatment regimens. It will be appreciated by thoseskilled in the art that body fluids used for diagnostic testing includewithout limitation blood, serum, plasma, urine, cerebrospinal fluid andother fluids. In a preferred embodiment the body fluid selected as thesample is blood, serum or plasma. The assay response, level,concentration or quantity of a nucleosome associated nucleotide in abody fluid may be expressed in absolute terms or relative terms, forexample without limitation as a proportion of the total nucleosome levelpresent or as a ratio to the level of nucleosomes containing anothernucleotide or histone variant or histone PTM or to the level of totalDNA.

In one embodiment of the invention the nucleosome associated nucleotidemeasurement is used as a member of a diagnostic panel of tests ormeasurements for the detection or diagnosis of the disease status of asubject including, without limitation, a clinical diagnosis of adisease, a differential diagnosis of disease type or subtype, or adisease prognosis, or a disease relapse, or a diagnosis of subjectsusceptibility to treatment regimens

As all or most circulating cell free DNA is reported to exist asnucleosome associated DNA, it will be clear to those skilled in the artthat diagnosis or detection of disease state can be achieved bydetection or measurement of nucleotides per se using a direct nucleotideimmunoassay of the invention with no DNA extraction step in a biologicalfluid, rather than, or in addition to, an immunoassay for nucleosomeassociated nucleotides. According to a further aspect of the inventionthere is provided a non-extraction nucleotide immunoassay method fordetecting or diagnosing the presence of a disease by measuring ordetecting the presence and/or the level or concentration of a nucleotidein a body fluid, and using the detected level as a biomarker (eitheralone as a member of a panel of tests) of the disease status of asubject including, without limitation, a clinical diagnosis of adisease, a differential diagnosis of disease type or subtype, or adisease prognosis, or a disease relapse, or a diagnosis of subjectsusceptibility to treatment regimens. It will be appreciated by thoseskilled in the art that body fluids used for diagnostic testing includewithout limitation blood, serum, plasma, urine, cerebrospinal fluid andother fluids. In a preferred embodiment the body fluid selected as thesample is blood, serum or plasma. The assay response, level,concentration or quantity of a nucleotide in a body fluid may beexpressed in absolute terms or relative terms, for example withoutlimitation as a proportion of the total nucleosome level present or as aratio to the level of another nucleotide or histone variant or histonePTM or to the level of total DNA.

According to a further aspect of the invention there is provided amethod for detecting or measuring the presence and/or the level ofnucleosomes containing a nucleotide in a cell which comprises the stepsof:

-   -   (i) isolating chromatin from a cell;    -   (ii) breaking down the chromatin to form mono-nucleosomes and/or        oligo-nucleosomes; and    -   (iii) detecting or measuring the presence of a nucleotide in the        mono-nucleosomes and/or oligo-nucleosomes by means of an        immunoassay method of the invention.

Methods for producing mono-nucleosomes and/or oligo-nucleosomes fromchromatin are well known in the art and include enzyme digestion andsonication (Dai et al, 2011). In one embodiment the nucleotide selectedfor detection by the method is a commonly occurring nucleotide thatoccurs in all or most intact nucleosomes, providing a method for thedetection or measurement of nucleosomes per se. In another embodimentthe nucleotide selected for detection by the method is a commonlyoccurring nucleotide that occurs in all or most intact nucleosomes,providing a method for the detection or measurement of nucleosome boundDNA.

It will be appreciated by those skilled in the art that the describedmethod of detecting nucleosome associated nucleotides in cells ortissues has advantages over currently used methods including IHC, ordetecting nucleotides in DNA extracted from cells by restrictiondigestion and nearest-neighbour analysis, or by fluorescent assays usingchloracetaldehyde, or by inverse determination by methylation of all CpGsites using DNA methyltransferase in conjunction with tritium-labeledS-adenosyl methionine to calculate the amount of unmethylated CpG, or bydigestion of DNA into single nucleotides for analysis byhigh-performance liquid chromatography, thin-layer chromatography, orliquid chromatography followed by mass spectroscopy. The level,concentration or quantity of a particular nucleosome associatednucleotide may be expressed in absolute terms or relative terms, forexample as a proportion of the total nucleosomes present or as a ratioto the total level of nucleosomes or to the level of nucleosomescontaining another nucleotide or histone variant or histone PTM, or tothe total level of DNA.

It will be clear to those skilled in the art that the terms antibody,binder or ligand in regard to any aspect of the invention is notlimiting but intended to include any binder capable of binding toparticular molecules or entities and that any suitable binder can beused in the method of the invention. It will also be clear that the termnucleosomes is intended to include mononucleosomes and oligonucleosomesand any such chromatin fragments that can be analysed in fluid media.

According to another aspect of the invention there is provided a kit fordetecting or measuring nucleosomes which comprises a ligand or binderspecific for the nucleotide or a component part thereof, or astructural/shape mimic of the nucleosome or component part thereof,together with instructions for use of the kit in accordance with any ofthe methods defined herein.

According to a further aspect of the invention there is provided a kitfor detecting or measuring nucleosomes containing a nucleotide whichcomprises a ligand or binder specific for the nucleotide or a componentpart thereof, or a structural/shape mimic of the nucleotide or componentpart thereof, together with instructions for use of the kit inaccordance with any of the methods defined herein.

According to another aspect of the invention there is provided a methodfor identifying a nucleosome associated nucleotide biomarker or anucleotide biomarker for detecting or diagnosing disease status inanimals or humans which comprises the steps of:

-   -   (i) detecting or measuring the level of cell free nucleosomes        containing a nucleotide in a body fluid of diseased subjects;    -   (ii) detecting or measuring the level of cell free nucleosomes        containing a nucleotide in a body fluid of control subjects; and    -   (iii) using the difference between the levels detected in        diseased and control subjects to identify whether a nucleotide        is useful as a biomarker for that disease.

It will be clear to those skilled in the art that the control subjectsmay be selected on a variety of basis which may include, for example,subjects known to be free of the disease or may be subjects with adifferent disease (for example; for the investigation of differentialdiagnosis).

According to a further aspect of the invention there is provided amethod for identifying a nucleosome associated nucleotide biomarker or anucleotide biomarker for assessing the prognosis of a diseased animal orhuman subject which comprises the steps of:

-   -   (i) detecting or measuring the level of cell free nucleosomes        containing a nucleotide in a body fluid of diseased subjects;        and    -   (ii) correlating the level of cell free nucleosomes containing a        nucleotide detected in a body fluid of diseased subjects with        the disease outcome of the subjects.

According to a further aspect of the invention there is provided amethod for identifying a nucleotide biomarker to be used for theselection of a treatment regimen for a diseased animal or human subjectin need of treatment which comprises the steps of:

-   -   (i) detecting or measuring the level of cell free nucleosomes        containing a nucleotide in a body fluid of diseased subjects;        and    -   (ii) correlating the level of cell free nucleosomes containing a        nucleotide detected in a body fluid of diseased subjects with        the observed efficacy of a treatment regimen in those subjects.

According to a further aspect of the invention there is provided amethod for identifying a nucleosome associated nucleotide biomarker or anucleotide biomarker to be used for monitoring the treatment of adiseased animal or human subject which comprises the steps of:

-   -   (i) detecting or measuring the level of cell free nucleosomes        containing a nucleotide in a body fluid of a diseased subject;    -   (ii) repeating said detection or measurement on one or more        occasions during the disease progression of the subject; and    -   (iii) correlating the level of cell free nucleosomes containing        a nucleotide detected in a body fluid of a diseased subject with        the disease progression in the subject.

According to a further aspect of the invention, there is provided abiomarker identified by the method as defined herein.

It is known in the art that one may detect the presence of a moiety thatis comprised as part of a complex containing other moieties byimmunoassay methods. It will be clear to those skilled in the art thatcell free nucleosomes containing a nucleotide can be detected in abiological fluid including blood, plasma, serum and urine by a procedureinvolving the direct immunoassay of the nucleotide itself in the fluid.In this procedure a single antibody immunoassay, utilising an antibodydirected to an epitope present on a nucleotide, or a 2-site immunoassay,utilising two antibodies directed to two epitopes present on anucleotide, is used to detect the presence of a nucleotide within anucleosome. Thus in another embodiment of the invention a nucleotidecontained within a nucleosome is detected directly in a biological fluidincluding blood, plasma, serum and urine by use of an immunoassay methodfor a nucleotide.

Thus in one embodiment of the invention a nucleosome associatednucleotide is detected directly without prior extraction in a biologicalfluid including blood, plasma, serum and urine using an immunoassay forthe nucleotide.

A further aspect of the invention provides ligands or binders, such asnaturally occurring or chemically synthesised compounds, capable ofspecific binding to the biomarker. A ligand or binder according to theinvention may comprise a peptide, an antibody or a fragment thereof, ora synthetic ligand such as a plastic antibody, or an aptamer oroligonucleotide, capable of specific binding to the biomarker. Theantibody can be a monoclonal antibody or a fragment thereof capable ofspecific binding to the biomarker. A ligand according to the inventionmay be labeled with a detectable marker, such as a luminescent,fluorescent, enzyme or radioactive marker; alternatively or additionallya ligand according to the invention may be labelled with an affinitytag, e.g. a biotin, avidin, streptavidin or His (e.g. hexa-His) tag.Alternatively ligand binding may be determined using a label-freetechnology for example that of ForteBio Inc.

A biosensor according to the invention may comprise the biomarker or astructural/shape mimic thereof capable of specific binding to anantibody against the biomarker. Also provided is an array comprising aligand or mimic as described herein.

Also provided by the invention is the use of one or more ligands asdescribed herein, which may be naturally occurring or chemicallysynthesised, and is suitably a peptide, antibody or fragment thereof,aptamer or oligonucleotide, or the use of a biosensor of the invention,or an array of the invention, or a kit of the invention to detect and/orquantify the biomarker. In these uses, the detection and/orquantification can be performed on a biological sample as definedherein.

Diagnostic or monitoring kits are provided for performing methods of theinvention. Such kits will suitably comprise a ligand according to theinvention, for detection and/or quantification of the biomarker, and/ora biosensor, and/or an array as described herein, optionally togetherwith instructions for use of the kit.

A further aspect of the invention is a kit for detecting the presence ofa disease state, comprising a biosensor capable of detecting and/orquantifying one or more of the biomarkers as defined herein.

Biomarkers for detecting the presence of a disease are essential targetsfor discovery of novel targets and drug molecules that retard or haltprogression of the disorder. As the level of the biomarker is indicativeof disorder and of drug response, the biomarker is useful foridentification of novel therapeutic compounds in in vitro and/or in vivoassays. Biomarkers of the invention can be employed in methods forscreening for compounds that modulate the activity of the biomarker.

Thus, in a further aspect of the invention, there is provided the use ofa binder or ligand, as described, which can be a peptide, antibody orfragment thereof or aptamer or oligonucleotide according to theinvention; or the use of a biosensor according to the invention, or anarray according to the invention; or a kit according to the invention,to identify a substance capable of promoting and/or of suppressing thegeneration of the biomarker.

Also there is provided a method of identifying a substance capable ofpromoting or suppressing the generation of the biomarker in a subject,comprising administering a test substance to a subject animal anddetecting and/or quantifying the level of the biomarker present in atest sample from the subject.

The term “biomarker” means a distinctive biological or biologicallyderived indicator of a process, event, or condition. Biomarkers can beused in methods of diagnosis, e.g. clinical screening, and prognosisassessment and in monitoring the results of therapy, identifyingpatients most likely to respond to a particular therapeutic treatment,drug screening and development. Biomarkers and uses thereof are valuablefor identification of new drug treatments and for discovery of newtargets for drug treatment.

The terms “detecting” and “diagnosing” as used herein encompassidentification, confirmation, and/or characterisation of a diseasestate. Methods of detecting, monitoring and of diagnosis according tothe invention are useful to confirm the existence of a disease, tomonitor development of the disease by assessing onset and progression,or to assess amelioration or regression of the disease. Methods ofdetecting, monitoring and of diagnosis are also useful in methods forassessment of clinical screening, prognosis, choice of therapy,evaluation of therapeutic benefit, i.e. for drug screening and drugdevelopment.

Efficient diagnosis and monitoring methods provide very powerful“patient solutions” with the potential for improved prognosis, byestablishing the correct diagnosis, allowing rapid identification of themost appropriate treatment (thus lessening unnecessary exposure toharmful drug side effects), and reducing relapse rates.

In one embodiment, said biomarker is released from the cells of atumour. Thus, according to a further aspect of the invention there isprovided a method for the detection of a tumour growth which comprisesthe steps of (i) measuring a biomarker in a biological sample that isassociated with or released from the cells of a tumour and (ii)demonstrating that the level of said biomarker is associated with thesize, stage, aggressiveness or dissemination of the tumour.

It is known that increased cell turnover, cell death and apoptosis leadto increased circulatory levels of cell free nucleosomes (Holdenriederet al, 2001). Circulating cell free nucleosomes level is a non-specificindicator and occurs in a variety of conditions including inflammatorydiseases, a large variety of benign and malignant conditions, autoimmunediseases, as well as following trauma or ischaemia (Holdenrieder et al2001). It will be clear to those skilled in the art that the inventionwill have application in a variety of disease areas where circulatingnucleosomes have been found in subjects. These include, withoutlimitation, trauma (for example; severe injury or surgery), extremeexercise (for example running a marathon), stroke and heart attack,sepsis or other serious infection and endometriosis. We have used theimmunoassay method of the invention to measure nucleosome levels andinvestigate their nucleotide and histone structure variability in avariety of such diseases including cardiomyopathy, systemic lupuserythematosus, ulcerative colitis, chronic obstructive pulmonarydisease, Crohn's disease and rheumatoid arthritis and compared thesewith the results of healthy subjects. We can detect nucleosomes anddetermine their relative structures (in terms of histone and nucleotidecomposition) in all these diseases. As methods of the current inventionare capable of detection of a wider range of nucleosomes than currentnucleosome ELISA methods, the methods of the invention have applicationsin a wide range of cancer and non-cancer disease areas.

The immunoassays of the invention include immunometric assays employingenzyme detection methods (for example ELISA), fluorescence labelledimmunometric assays, time-resolved fluorescence labelled immunometricassays, chemiluminescent immunometric assays, immunoturbidimetricassays, particulate labelled immunometric assays and immunoradiometricassays and competitive immunoassay methods including labelled antigenand labelled antibody competitive immunoassay methods with a variety oflabel types including radioactive, enzyme, fluorescent, time-resolvedfluorescent and particulate labels. All of said immunoassay methods arewell known in the art, see for example Salgame et al, 1997 and vanNieuwenhuijze et al, 2003.

In one embodiment, said biological sample comprises a body fluid. Forexample, biological samples that may be tested in a method of theinvention include cerebrospinal fluid (CSF), whole blood, blood serum,plasma, menstrual blood, endometrial fluid, urine, saliva, or otherbodily fluid (stool, tear fluid, synovial fluid, sputum), breath, e.g.as condensed breath, or an extract or purification therefrom, ordilution thereof. Biological samples also include specimens from a livesubject, or taken post-mortem. The samples can be prepared, for examplewhere appropriate diluted or concentrated, and stored in the usualmanner.

In one embodiment, the method of the invention is repeated on multipleoccasions. This embodiment provides the advantage of allowing thedetection results to be monitored over a time period. Such anarrangement will provide the benefit of monitoring or assessing theefficacy of treatment of a disease state. Such monitoring methods of theinvention can be used to monitor onset, progression, stabilisation,amelioration, relapse and/or remission.

Thus, the invention also provides a method of monitoring efficacy of atherapy for a disease state in a subject, suspected of having such adisease, comprising detecting and/or quantifying the biomarker presentin a biological sample from said subject. In monitoring methods, testsamples may be taken on two or more occasions. The method may furthercomprise comparing the level of the biomarker(s) present in the testsample with one or more control(s) and/or with one or more previous testsample(s) taken earlier from the same test subject, e.g. prior tocommencement of therapy, and/or from the same test subject at an earlierstage of therapy. The method may comprise detecting a change in thenature or amount of the biomarker(s) in test samples taken on differentoccasions.

Thus, according to a further aspect of the invention, there is provideda method for monitoring efficacy of therapy for a disease state in ahuman or animal subject, comprising:

-   -   (i) quantifying the amount of the biomarker as defined herein;        and    -   (ii) comparing the amount of said biomarker in a test sample        with the amount present in one or more control(s) and/or one or        more previous test sample(s) taken at an earlier time from the        same test subject.

A change in the level of the biomarker in the test sample relative tothe level in a previous test sample taken earlier from the same testsubject may be indicative of a beneficial effect, e.g. stabilisation orimprovement, of said therapy on the disorder or suspected disorder.Furthermore, once treatment has been completed, the method of theinvention may be periodically repeated in order to monitor for therecurrence of a disease.

Methods for monitoring efficacy of a therapy can be used to monitor thetherapeutic effectiveness of existing therapies and new therapies inhuman subjects and in non-human animals (e.g. in animal models). Thesemonitoring methods can be incorporated into screens for new drugsubstances and combinations of substances.

In a further embodiment the monitoring of more rapid changes due to fastacting therapies may be conducted at shorter intervals of hours or days.

According to a further aspect of the invention, there is provided amethod for identifying a biomarker for detecting the presence of adisease state. The term “identifying” as used herein means confirmingthe presence of the biomarker present in the biological sample.Quantifying the amount of the biomarker present in a sample may includedetermining the concentration of the biomarker present in the sample.Identifying and/or quantifying may be performed directly on the sample,or indirectly on an extract therefrom, or on a dilution thereof.

In alternative aspects of the invention, the presence of the biomarkeris assessed by detecting and/or quantifying antibody or fragmentsthereof capable of specific binding to the biomarker that are generatedby the subject's body in response to the biomarker and thus are presentin a biological sample from a subject having a disease state.

Identifying and/or quantifying can be performed by any method suitableto identify the presence and/or amount of a specific protein in abiological sample from a patient or a purification or extract of abiological sample or a dilution thereof. In methods of the invention,quantifying may be performed by measuring the concentration of thebiomarker in the sample or samples. Biological samples that may betested in a method of the invention include those as definedhereinbefore. The samples can be prepared, for example where appropriatediluted or concentrated, and stored in the usual manner.

Identification and/or quantification of biomarkers may be performed bydetection of the biomarker or of a fragment thereof, e.g. a fragmentwith C-terminal truncation, or with N-terminal truncation. Fragments aresuitably greater than 4 amino acids in length, for example 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length.It is noted in particular that peptides of the same or related sequenceto that of histone tails are particularly useful fragments of histoneproteins.

The biomarker may be directly detected, e.g. by SELDI or MALDI-TOF.Alternatively, the biomarker may be detected directly or indirectly viainteraction with a ligand or ligands such as an antibody or abiomarker-binding fragment thereof, or other peptide, or ligand, e.g.aptamer, or oligonucleotide, capable of specifically binding thebiomarker. The ligand or binder may possess a detectable label, such asa luminescent, fluorescent or radioactive label, and/or an affinity tag.

For example, detecting and/or quantifying can be performed by one ormore method(s) selected from the group consisting of: SELDI (-TOF),MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Massspec (MS), reverse phase (RP) LC, size permeation (gel filtration), ionexchange, affinity, HPLC, UPLC and other LC or LC MS-based techniques.Appropriate LC MS techniques include ICAT® (Applied Biosystems, CA,USA), or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography(e.g. high pressure liquid chromatography (HPLC) or low pressure liquidchromatography (LPLC)), thin-layer chromatography, NMR (nuclear magneticresonance) spectroscopy could also be used.

Methods of diagnosing or monitoring according to the invention maycomprise analysing a sample by SELDI TOF or MALDI TOF to detect thepresence or level of the biomarker. These methods are also suitable forclinical screening, prognosis, monitoring the results of therapy,identifying patients most likely to respond to a particular therapeutictreatment, for drug screening and development, and identification of newtargets for drug treatment.

Identifying and/or quantifying the analyte biomarkers may be performedusing an immunological method, involving an antibody, or a fragmentthereof capable of specific binding to the biomarker. Suitableimmunological methods include sandwich immunoassays, such as sandwichELISA, in which the detection of the analyte biomarkers is performedusing two antibodies which recognize different epitopes on a analytebiomarker; radioimmunoassays (RIA), direct, indirect or competitiveenzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA),Fluorescence immunoassays (FIA), western blotting, immunoprecipitationand any particle-based immunoassay (e.g. using gold, silver, or latexparticles, magnetic particles, or Q-dots). Immunological methods may beperformed, for example, in microtitre plate or strip format.

In one embodiment, one or more of the biomarkers may be replaced by amolecule, or a measurable fragment of the molecule, found upstream ordownstream of the biomarker in a biological pathway.

The identification of key biomarkers specific to a disease is central tointegration of diagnostic procedures and therapeutic regimes. Usingpredictive biomarkers appropriate diagnostic tools such as biosensorscan be developed; accordingly, in methods and uses of the invention,identifying and quantifying can be performed using a biosensor,microanalytical system, microengineered system, microseparation system,immunochromatography system or other suitable analytical devices. Thebiosensor may incorporate an immunological method for detection of thebiomarker(s), electrical, thermal, magnetic, optical (e.g. hologram) oracoustic technologies. Using such biosensors, it is possible to detectthe target biomarker(s) at the anticipated concentrations found inbiological samples.

As used herein, the term “biosensor” means anything capable of detectingthe presence of the biomarker. Examples of biosensors are describedherein.

Biosensors according to the invention may comprise a ligand binder orligands, as described herein, capable of specific binding to thebiomarker. Such biosensors are useful in detecting and/or quantifying abiomarker of the invention.

The biomarker(s) of the invention can be detected using a biosensorincorporating technologies based on “smart” holograms, or high frequencyacoustic systems, such systems are particularly amenable to “bar code”or array configurations.

In smart hologram sensors (Smart Holograms Ltd, Cambridge, UK), aholographic image is stored in a thin polymer film that is sensitised toreact specifically with the biomarker. On exposure, the biomarker reactswith the polymer leading to an alteration in the image displayed by thehologram. The test result read-out can be a change in the opticalbrightness, image, colour and/or position of the image. For qualitativeand semi-quantitative applications, a sensor hologram can be read byeye, thus removing the need for detection equipment. A simple coloursensor can be used to read the signal when quantitative measurements arerequired. Opacity or colour of the sample does not interfere withoperation of the sensor. The format of the sensor allows multiplexingfor simultaneous detection of several substances. Reversible andirreversible sensors can be designed to meet different requirements, andcontinuous monitoring of a particular biomarker of interest is feasible.

Suitably, biosensors for detection of one or more biomarkers of theinvention combine biomolecular recognition with appropriate means toconvert detection of the presence, or quantitation, of the biomarker inthe sample into a signal. Biosensors can be adapted for “alternate site”diagnostic testing, e.g. in the ward, outpatients' department, surgery,home, field and workplace.

Biosensors to detect one or more biomarkers of the invention includeacoustic, plasmon resonance, holographic, Bio-Layer Interferometry (BLI)and microengineered sensors. Imprinted recognition elements, thin filmtransistor technology, magnetic acoustic resonator devices and othernovel acousto-electrical systems may be employed in biosensors fordetection of the one or more biomarkers of the invention.

Methods involving identification and/or quantification of one or morebiomarkers of the invention can be performed on bench-top instruments,or can be incorporated onto disposable, diagnostic or monitoringplatforms that can be used in a non-laboratory environment, e.g. in thephysician's office or at the patient's bedside. Suitable biosensors forperforming methods of the invention include “credit” cards with opticalor acoustic readers. Biosensors can be configured to allow the datacollected to be electronically transmitted to the physician forinterpretation and thus can form the basis for e-medicine.

Diagnostic kits for the diagnosis and monitoring of the presence of adisease state are described herein. In one embodiment, the kitsadditionally contain a biosensor capable of identifying and/orquantifying a biomarker. Suitably a kit according to the invention maycontain one or more components selected from the group: a ligand binder,or ligands, specific for the biomarker or a structural/shape mimic ofthe biomarker, one or more controls, one or more reagents and one ormore consumables; optionally together with instructions for use of thekit in accordance with any of the methods defined herein.

The identification of biomarkers for a disease state permits integrationof diagnostic procedures and therapeutic regimes. Detection of abiomarker of the invention can be used to screen subjects prior to theirparticipation in clinical trials. The biomarkers provide the means toindicate therapeutic response, failure to respond, unfavourableside-effect profile, degree of medication compliance and achievement ofadequate serum drug levels. The biomarkers may be used to providewarning of adverse drug response. Biomarkers are useful in developmentof personalized therapies, as assessment of response can be used tofine-tune dosage, minimise the number of prescribed medications, reducethe delay in attaining effective therapy and avoid adverse drugreactions. Thus by monitoring a biomarker of the invention, patient carecan be tailored precisely to match the needs determined by the disorderand the pharmacogenomic profile of the patient, the biomarker can thusbe used to titrate the optimal dose, predict a positive therapeuticresponse and identify those patients at high risk of severe sideeffects.

Biomarker-based tests provide a first line assessment of ‘new’ patients,and provide objective measures for accurate and rapid diagnosis, notachievable using the current measures.

Furthermore, diagnostic biomarker tests are useful to identify familymembers or patients with mild or asymptomatic disease or who may be athigh risk of developing symptomatic disease. This permits initiation ofappropriate therapy, or preventive measures, e.g. managing risk factors.These approaches are recognised to improve outcome and may prevent overtonset of the disorder.

Biomarker monitoring methods, biosensors and kits are also vital aspatient monitoring tools, to enable the physician to determine whetherrelapse is due to worsening of the disorder. If pharmacologicaltreatment is assessed to be inadequate, then therapy can be reinstatedor increased; a change in therapy can be given if appropriate. As thebiomarkers are sensitive to the state of the disorder, they provide anindication of the impact of drug therapy.

The invention will now be illustrated with reference to the followingnon-limiting examples.

EXAMPLE 1

A commercially available nucleosome preparation produced by digestion ofchromatin extracted from MCF7 cells in which the DNA and proteins in thenucleosome are cross-linked for stability (ensuring that all histonespresent in the preparation are incorporated into intact nucleosomes) wasassayed for methylated DNA using an ELISA method for the nucleosomeassociated nucleotide 5-methylcytosine using a solid phase anti-histonecapture antibody that binds intact nucleosomes and a biotinylatedmonoclonal anti-5-methylcytosine detection antibody. The nucleosomesample was serially diluted in fetal calf serum and was tested induplicate in the ELISA. Neat fetal calf serum was also run in the ELISAas a control sample containing no cell free nucleosomes. The assaymethod was as follows: A solution of anti-histone antibody in 0.1Mphosphate buffer pH 7.4 was added to microtitre wells (100 μL/well) andincubated overnight at 4° C. to coat the wells with capture antibody.Excess anti-histone antibody was decanted. A solution of bovine serumalbumin (20 g/L) was added to the wells (200 μL/well) and incubated 30minutes at room temperature to block excess protein binding sites on thewells. Excess bovine serum albumin solution was decanted and the wellswere washed three times with wash buffer (200 μL/well, 0.05M TRIS/HClbuffer pH 7.5 containing 1% Tween 20). Sample (10 μL/well) and assaybuffer (50 μL/well, 0.05M TRIS/HCl pH 7.5 containing 0.9% NaCl, 0.05%sodium deoxycholate and 1% Nonidet P40 substitute) were added to thewells incubated 90 minutes at room temperature with mild agitation. Thesample and assay buffer mixture was decanted and the wells were washedthree times with wash buffer (200 μL/well). A solution of biotinylatedanti-5-methylcytosine detection antibody was added (50 μL/well) andincubated 90 minutes at room temperature with mild agitation. Excessdetection antibody was decanted and the wells were again washed threetimes with wash buffer (200 μL/well). A solution containing astreptavidin-horse radish peroxidase conjugate was added (50 μL/well)and incubated 30 minutes at room temperature with mild agitation. Excessconjugate was decanted and the wells were again washed three times withwash buffer (200 μL/well). A coloured substrate solution (100 μL/well,2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt)was added and incubated 20 minutes at room temperature with mildagitation. The optical density (OD) of the wells was measured at awavelength of 405 nm using a standard microtitre plate reader. A doseresponse curve of increasing colour with increasing nucleosomeassociated anti-5-methylcytosine concentration was observed with a lowbackground signal observed in the absence of 5-methylcytosine (fetalcalf serum). The positive ELISA signal indicates that the5-methylcytosine detected by the ELISA is incorporated within an intactnucleosome comprising both histone protein and DNA as (i) the captureantibody binds to histones in the sample and (ii) detection antibodybinds to the 5-methylcytosine component of DNA. The results are shown inFIG. 1.

EXAMPLE 2

A commercially available nucleosome preparation produced by digestion ofchromatin extracted from A375 cells in which the DNA and proteins in thenucleosome are cross-linked for stability (ensuring that all histonespresent in the preparation are incorporated into intact nucleosomes) wasassayed for 5-hydroxymethylated DNA using an ELISA method for thenucleosome associated nucleotide 5-hydroxymethylcytosine using a solidphase anti-histone capture antibody that binds intact nucleosomes and abiotinylated monoclonal anti-5-hydroxymethylcytosine detection antibody.The nucleosome sample was serially diluted in fetal calf serum and wastested in duplicate in the ELISA. Neat fetal calf serum was also run inthe ELISA as a control sample containing no cell free nucleosomes. Theassay method was as follows: A solution of anti-histone antibody in 0.1Mphosphate buffer pH 7.4 was added to microtitre wells (100 μL/well) andincubated overnight at 4° C. to coat the wells with capture antibody.Excess anti-histone antibody was decanted. A solution of bovine serumalbumin (20 g/L) was added to the wells (200 μL/well) and incubated 30minutes at room temperature to block excess protein binding sites on thewells. Excess bovine serum albumin solution was decanted and the wellswere washed three times with wash buffer (200 μL/well, 0.05M TRIS/HClbuffer pH 7.5 containing 1% Tween 20). Sample (10 μL/well) and assaybuffer (50 μL/well, 0.05M TRIS/HCl pH 7.5 containing 0.9% NaCl, 0.05%sodium deoxycholate and 1% Nonidet P40 substitute) were added to thewells incubated 90 minutes at room temperature with mild agitation. Thesample and assay buffer mixture was decanted and the wells were washedthree times with wash buffer (200 μL/well). A solution of biotinylatedanti-5-hydroxymethylcytosine detection antibody was added (50 μL/well)and incubated 90 minutes at room temperature with mild agitation. Excessdetection antibody was decanted and the wells were again washed threetimes with wash buffer (200 μL/well). A solution containing astreptavidin-horse radish peroxidase conjugate was added (50 μL/well)and incubated 30 minutes at room temperature with mild agitation. Excessconjugate was decanted and the wells were again washed three times withwash buffer (200 μL/well). A coloured substrate solution (100 μL/well,2,2′-Azinobis [3-ethylbenzothiazoline-6-sulfonic acid] -diammonium salt)was added and incubated 20 minutes at room temperature with mildagitation. The optical density (OD) of the wells was measured at awavelength of 405 nm using a standard microtitre plate reader. A doseresponse curve of increasing colour with increasing nucleosomeassociated 5-hydroxymethylcytosine concentration was observed with a lowbackground signal observed in the absence of 5-hydroxymethylcytosine(fetal calf serum). The positive ELISA signal indicates that the5-hydroxymethylcytosine detected by the ELISA is incorporated within anintact nucleosome comprising both histone protein and DNA as (i) thecapture antibody binds to histones in the sample and (ii) detectionantibody binds to the 5-hydroxymethylcytosine component of DNA. Theresults are shown in FIG. 2.

EXAMPLE 3

We used two nucleosome ELISA methods of the current art to measure thecirculating cell free nucleosome content of serum and plasma bloodsamples taken from 20 healthy subjects. The first current ELISA method(ELISA 1) was the Roche Cell Death ELISA and the other (ELISA 2) anELISA employing an anti-histone capture antibody and an anti-histone-DNAcomplex detection antibody. The nucleosome levels detected by bothcurrent nucleosome ELISA methods were lower in normal plasma than innormal serum. The normal range (expressed in optical density units) forthe serum level of nucleosomes was calculated (mean±2 standarddeviations of the mean of the 20 healthy subject serum results) to be0-4.3 OD units for ELISA 1 and 0-1.4 for ELISA 2. The respective rangesfor plasma were 0-0.95 and 0-0.96. The results are shown in FIG. 3.

We also measured the levels of nucleosomes containing the two nucleosomeassociated nucleotides as well as 3 nucleosome associated histonevariants and a histone PTM in the same 20 samples taken from healthysubjects. The results show that the healthy serum samples have uniformlylow levels of nucleosomes containing histone variants or PTM ornucleotides. The normal ranges (expressed as optical density) for theserum level of nucleosomes containing histone variants, PTM ornucleotides were; (a) 0-0.36 for mH2A1.1, (b) 0.05-0.78 for mH2A2, (c)0.11-0.58 for H2AZ, (d) 0.06-0.61 for P-H2AX(Ser139), (e) 0.06-0.36 for5-methylcytosine and (f) 0.03-0.36 for 5-hydroxymethylcytrosine. Themeasured EDTA plasma results were higher for all 20 healthy subjects.The results are shown in FIGS. 4, 5, 6, 7, 8 and 9.

EXAMPLE 4

We measured cell free nucleosomes containing 5-methylcytosine in EDTAplasma taken from 13 healthy subjects and 55 subjects with cancer of thestomach, large intestine, rectum, lung (small cell carcinoma and variousnon-small cell carcinomas), breast, ovary, pancreas, prostate, kidneyand various oral cancers (oral cavity, palate, pharynx and larynx). Allof the 13 samples from healthy subjects were positive for one or morecell free nucleosome type. All of the 55 samples from cancer patientswere positive for all the cell free nucleosome types assayed. However,the levels detected in samples taken from cancer subjects were higherthan found in samples from healthy subjects and the results showed thathealthy and cancer subjects can be discriminated. For example the normalrange calculated in OD terms as the mean±2 standard deviations of themean, for nucleosome associated 5-methylcytosine 0-1.41. Using thiscut-off value all 13 healthy samples were negative and 30 of the 55cancer samples were positive (an overall clinical sensitivity of 55%)including 38% (3 of 8) of stomach, 60% (3 of 5) of large intestinal, 33%(1 of 3) of rectal, 33% (2 of 6) of small cell lung, 64% (9 of 14) ofnon-small cell lung, 33% (2 of 6) of breast, 100% (1 of 1) of ovarian,100% (1 of 1) of pancreas, 33% (2 of 6) of prostate, 100% (1 of 1) ofkidney and 60% (3 of 5) of oral cancer samples. The results are shown inFIG. 17.

We also used the methods of the invention to measure a variety of othernucleosome associated structures in the same samples. The results ofthese immunoassays were compiled to provide a profile of nucleosomestructures in samples taken from cancer patients normalised relative todetected levels of nucleosomes containing 5-methylcytosine. We comparedthe resulting profiles to the nucleosome structure of samples taken fromhealthy subjects. The nucleosome structure profile of cell freenucleosomes was found to be different to those of healthy subjects. Theresults are shown in FIG. 20. We similarly compiled nucleosome structureprofiles for samples taken from a variety of non-cancer diseases andcompared these to the profile of nucleosomes in samples taken fromcancer patients and from healthy subjects. The results are shown in FIG.21.

We then performed another similar experiment including samples from 10healthy subjects and a further 62 patients with cancer of various types.The results were similar to the first experiment. For example using theresults for nucleosome associated 5-methylcytosine and a cut-off ofmean+2 standard deviations of the mean of the results for healthysubjects, negative results were obtained for all 10 healthy subjects andpositive results were obtained for 95% (61 of 62) of cancer patientsincluding 9 of 9 prostate cancer patients, 5 of 5 skin cancer patients,8 of 8 esophagus cancer patients, 12 of 13 bladder cancer patients, 2 of2 cervix cancer patients and 1 of 1 colon cancer patients, 4 of 4 breastcancer patients, 7 of 7 ovary cancer patients, 7 of 7 larynx cancerpatients, 3 of 3 lung cancer patients and 3 of 3 renal cancer patients.The results are shown in FIG. 18. This result indicates that serumnucleotide levels and nucleosome associated nucleotides levels,including particularly 5-methylcytosine, are clinically sensitivebiomarkers for cancer.

EXAMPLE 5

We used two nucleosome ELISA methods of the current art to measure thecirculating cell free nucleosome content of samples taken from 3subjects with colon cancer, 13 subjects with lung cancer, 2 subjectswith pancreatic cancer, 1 subject with oral cancer and a nucleosomesample produced from healthy subjects according to the method ofHoldenrieder (*Holdenrieder et al, 2001). The first current ELISA method(ELISA 1) was the Roche Cell Death ELISA and the other (ELISA 2) anELISA employing an anti-histone capture antibody and an anti-histone-DNAcomplex detection antibody.

We also measured the levels of nucleosomes containing the nucleotides5-methylcytosine and 5-hydroxymethylcytosine as a well as three varianthistones and a histone PTM in the same 19 samples taken from cancersubjects. The results show that, although low nucleosome results forELISA methods of the current art were detected for most subjects,particularly for pancreatic and oral cancer patients, most of thesesamples have higher detectable levels of nucleosomes that contain one ormore nucleosome associated nucleotides or variant histones. The resultsfor samples taken from 3 subjects with colon cancer, 13 subjects withlung cancer, 2 subjects with pancreatic cancer and 1 subject with oralcancer are shown in FIGS. 10, 11, 12, and 13 respectively. Significantnucleosome associated histone variant levels and histone PTM levels weredetected in 16 of the 19 cancer samples (all but 3 lung cancer samples).In addition significant nucleosome associated 5-hydroxymethylcytosinelevels were detected in 12 of the 19 cancer samples. Furthermore,significant nucleosome associated 5-methylcytosine levels were detectedin all 19 cancer samples.

Furthermore the pattern of nucleosome levels containing differentnucleotide, histone variant and histone PTM levels is not uniform forall subjects but displays different patterns for different cancerstested. To facilitate comparison between results for subjects with thesame or different cancers; the results for the nucleosome tests (fornucleosomes containing macroH2A1.1, macroH2A2, H2AZ, P-H2AX(Ser139),5-methylcytosine, 5-hydroxymethylcytosine) were normalised as aproportion of the OD signal observed for nucleosomes containing5-methylcytosine. The normalised results (with error bars showing thestandard deviation in results where samples from more than one subjectwere tested) are shown for each cancer in FIG. 14 as well as the sameresults for the nucleosome sample produced from healthy subjects (mH2A2and 5-hydroxymethylcytosine were not measured for this sample). FIG. 14shows that the distribution pattern of nucleosomes containing thedifferent normalised nucleotides, histone variants or PTM in all fourcancers investigated differs quite markedly to the distribution ofnucleosomes in the sample prepared from healthy subjects. For examplethe relative level of nucleosomes containing macroH2A1.1 in the healthynucleosome sample differs from that detected in the samples of any ofthe cancer types. Thus the present invention can be used as a method forthe detection of cancer in a simple blood based screening test. It willbe clear to those skilled in the art that the invention includes thetesting of nucleosomes containing other further nucleotides and/orhistone variants and/or histone modifications to further or betterdiscriminate between circulating cell free nucleosomes of tumour orother disease origin.

Furthermore the pattern of nucleosome types observed differs fordifferent cancer types. For example; the samples taken from subjectswith colon, pancreatic and oral cancer can be distinguished by differentnormalised levels of nucleosome associated H2AZ and5-hydroxymethylcytosine. Similarly oral cancer has different normalisedlevels of both nucleosomes containing mH2A2 or P-H2AX(Ser139) than anyof the other three cancer types and samples from subjects withpancreatic cancer can be distinguished from samples from subjects withcolon cancer on the basis of a different relative level of nucleosomescontaining variant macroH2A1.1. Thus the present invention can be usedas a method to diagnose cancer generally and to distinguish a particularcancer type. It will be clear to those skilled in the art that theinvention includes the testing of nucleosomes containing other furtherhistone variants and/or histone modifications and/or nucleotides tofurther or better discriminate between circulating cell free nucleosomesof different specific tumour origin or other disease origin.

EXAMPLE 6

We tested the method of the invention in serum samples taken from 3healthy subjects and from 10 colon cancer patients. We measurednucleosomes containing 5-methylcytosine in these samples and the cancerresults were uniformly elevated over the results obtained for healthysubjects as shown in FIG. 16.

EXAMPLE 7

We measured the nucleosome associated 5-methylcytosine levels of humanEDTA plasma samples taken from lung and colon cancer patients. Thelevels detected were correlated with the disease progression of thepatients. The results shown in FIG. 19 indicate that nucleosomeassociated 5-methylcytosine levels increase with severity of disease interms of size, stage, nodal spread and nucleosome associated5-methylcytosine levels may be used, alone or as part of a diagnosticpanel, as indicators of disease progression.

REFERENCES

-   Allen et al, A simple method for estimating global DNA methylation    using bisulfite PCR of repetitive DNA elements. Nucleic Acids    Research: 32(3) e38DOI: 10.1093/nar/gnh032 Bawden et al, Detection    of histone modification in cell-free nucleosomes. WO 2005/019826,    2005-   Boulard et al, Histone variant macroH2A1 deletion in mice causes    female-specific steatosis. Epigenetics & Chromatin: 3(8), 1-13, 2010    Cell Biolabs, Inc. Product Manual for “Global DNA Methylation ELISA    Kit (5′-methyl-2′-deoxycytidine Quantitation”, 2011-   Dai et al, Detection of Post-translational Modifications on Native    Intact Nucleosomes by ELISA. http://www.jove.com/details.php?id=2593    doi: 10.3791/2593. J Vis Exp. 50 (2011).-   Deligezer et al, Sequence-Specific Histone Methylation Is Detectable    on Circulating Nucleosomes in Plasma. Clinical Chemistry 54(7),    1125-1131, 2008-   Epigentek Group Inc, Methylamp™ Global DNA Methylation    Quantification Kit, User Guide, Version 2.0802, 2009-   Esteller, Cancer epigenomics: DNA methylomes and    histone-modification maps Nature Reviews Genetics: 8, 286-298, 2007-   Feinberg and Vogelstein, Hypomethylation distinguishes genes of some    human cancers from their normal counterparts. Nature: 301, 89-92,    1983-   Grutzmann et al, Sensitive Detection of Colorectal Cancer in    Peripheral Blood by Septin 9 DNA Methylation Assay. PLoS ONE 3(11):    e3759. doi:10.1371/journal.pone.0003759, 2008-   Hervouet et al, Disruption of Dnmt1/PCNA/UHRF1 Interactions Promotes    Tumorigenesis from Human and Mice Glial Cells PLoS ONE 5(6): e11333.    doi:10.1371/journal.pone.0011333, 2010-   Hua et al, Genomic analysis of estrogen cascade reveals histone    variant H2A.Z associated with breast cancer progression. Molecular    Systems Biology 4; Article number 188; doi:10.1038/msb.2008.25, 2008-   Herranz and Esteller, DNA methylation and histone modifications in    patients with cancer: potential prognostic and therapeutic targets.    Methods Mol Biol.361:25-62, 2007-   Holdenrieder et al, Nucleosomes in serum of patients with benign and    malignant diseases. Int. J. Cancer (Pred. Oncol.): 95, 114-120, 2001-   *Holdenrieder et al, Nucleosomes in Serum as a Marker for Cell    Death. Clin Chem Lab Med; 39(7), 596-605, 2001-   Holdenrieder et al, Cell-Free DNA in Serum and Plasma: Comparison of    ELISA and Quantitative PCR. Clinical Chemistry: 51(8), 1544-1546,    2005-   Holdenreider and Stieber, Clinical use of circulating nucleosomes.    Critical Reviews in Clinical Laboratory Sciences; 46(1): 1-24, 2009-   Kapoor et al, The histone variant macroH2A suppresses melanoma    progression through regulation of CDK8. Nature: 468, 1105-1111, 2010-   Mansour et al, The Prognostic Significance of Whole Blood Global and    Specific DNA Methylation Levels in Gastric Adenocarcinoma. PLoS ONE    5(12): e15585. doi:10.1371/journal.pone.0015585, 2010-   Moore et al, Genomic DNA hypomethylation as a biomarker for bladder    cancer susceptibility in the Spanish Bladder Cancer Study: a    case-control study. The Lancet Oncology: 9(4), 359-366, 2008-   Ogoshi et al, Genome-wide profiling of DNA methylation in human    cancer cells. Genomics: In Press, 2011-   Pennings et al, DNA methylation, nucleosome formation and    positioning. Briefings in functional genomics and proteomics: 3(4),    351-361, 2005-   Rodriguez-Paredes and Esteller, Cancer epigenetics reaches    mainstream oncology. Nature Medicine: 17(3), 330-339, 2011-   Salgame et al, An ELISA for detection of apoptosis. Nucleic Acids    Research, 25(3), 680-681, 1997-   Sporn et al, Histone macroH2A isoforms predict the risk of lung    cancer recurrence. Oncogene: 28(38), 3423-8, 2009-   Stroud et al, 5-Hydroxymethylcytosine is associated with enhancers    and gene bodies in human embryonic stem cells. Genome Biology:    12:R54, 2011-   Tachiwana et al, Structures of human nucleosomes containing major    histone H3 variants. Acta Cryst: D67, 578-583, 2011-   Ting Hsiung et al, Global DNA Methylation Level in Whole Blood as a    Biomarker in Head and Neck Squamous Cell Carcinoma. Cancer    Epidemiology, Biomarkers & Prevention: 16(1), 108-114, 2007-   van Nieuwenhuijze et al, Time between onset of apoptosis and release    of nucleosomes from apoptotic cells: putative implications for    sysytemic lupus erythematosus. Ann Rheum Dis; 62: 10-14, 2003-   Vasser et al, Measurement of Global DNA Methylation. Genetic    Engineering and Biotechnology News: 29(7), 2009-   Whittle et al, The Genomic Distribution and Function of Histone    Variant HTZ-1 during C. elegans Embryogenesis. PLoS Genet 4(9):    1-17, 2008-   Zee et al, Global turnover of histone post-translational    modifications and variants in human cells Epigenetics & Chromatin.    3(22): 1-11, 2010-   Zhang et al, Analysis of global DNA methylation by hydrophilic    interaction ultra high-pressure liquid chromatography tandem mass    spectrometry. Analytical Biochemistry: 413(2), 164-170, 2011

It is claimed:
 1. A method for detecting cell-free nucleosomes with a5-methylcytosine or a 5-hydroxymethylcytosine DNA base in a blood, serumor plasma sample which comprises cell-free nucleosomes, comprising:contacting the blood, serum or plasma sample with a first binding agentwhich binds to the cell free nucleosomes and a second binding agentwhich binds to a 5-methylcytosine or a 5-hydroxymethylcytosine DNA base;and detecting cell-free nucleosomes with a 5-methylcytosine or5-hydroxymethylcytosine DNA base that bind to the first binding agentand the second binding agent.
 2. The method of claim 1, wherein thefirst binding agent is an antibody.
 3. The method of claim 2, whereinthe second binding agent is an antibody.
 4. The method of claim 1,wherein the second binding agent is an antibody.
 5. The method of claim1, wherein the sample is serum.
 6. The method of claim 1, wherein thesample is plasma.
 7. A method to discriminate a blood, serum or plasmasample as from a diseased subject or a healthy subject, comprising:providing a blood, serum or plasma sample from the subject; contactingthe sample with a first binding agent which binds to cell freenucleosomes and with a second binding agent which binds to a5-methylcytosine or a 5-hydroxymethylcytosine DNA base; and detectingcell-free nucleosomes with a 5-methylcytosine or 5-hydroxymethylcytosineDNA base that bind to the first binding agent and the second bindingagent to discriminate the sample as from a healthy subject or a diseasedsubject.
 8. The method of claim 7, wherein the first binding agent is anantibody.
 9. The method of claim 8, wherein the second binding agent isan antibody.
 10. The method of claim 7, wherein the second binding agentis an antibody.
 11. The method of claim 7, wherein the sample is serum.12. The method of claim 7, wherein the sample is plasma.
 13. The methodof claim 7, wherein said detecting discriminates the sample as from asubject with a disease selected from the group consisting of cancer,cardiomyopathy, systemic lupus erythematosus, colitis, chronicobstructive pulmonary disorder, Crohn's disease and rheumatoidarthritis.
 14. A method for detecting or diagnosing a disease status ina subject, comprising: (i) detecting or measuring a level or type ofcell free nucleosomes containing a particular DNA base, nucleotide ornucleoside in a body fluid of the subject; (ii) comparing the detectingor measuring of step (i) to the level or type of cell free nucleosomescontaining the particular DNA base, nucleotide or nucleoside in a bodyfluid of a healthy subject or a control subject; and (iii) using thedifference between the levels or types detected in diseased and controlsubjects to detect or identify disease status in the subject.
 15. A kitdetecting cell-free nucleosomes with a 5-methylcytosine or a5-hydroxymethylcytosine DNA base in a blood, serum or plasma samplewhich comprises cell-free nucleosomes, comprising: a first binding agentspecific for the 5-methylcytosine or a 5-hydroxymethylcytosine DNA base,a second binding agent which binds to cell free nucleosomes; andinstructions for use.